Cerebral Microcirculation Change And Astrocyte-mediated Regulation After Experimental Subarachnoid Hemorrhage In Rats

BACKGROUND: Cerebral vasospasm (CVS) is a common but fatal complication after subarachnoid hemorrhage (SAH). Delayed ischemic neurological dysfunction (DIND) is major cause of death and disable in patients with aneurysm. Most researches on CVS concentrated to the morphologic change of major arteries, and the main pathologic changes were believed to be the stenosis and wall thickening of the vessels induced by blood and hemolysate. Not all clinical findings can be well explained by CVS of major arteries, such as ischaemia without CVS, or CVS without ischaemia, the real determinant factors may relate to cerebral microcirculation. Relative few studies focused on the change of cerebral microcirculation after SAH, and conclusions were in discrepancies. There were opposite findings on whether the arterioles were constricted or dilated after SAH, and the precise course of cerebral microcirculation change after SAH and the modulation mechanism are not clear. Recent researches found that astrocyte(AS) has important role in modulating cerebral blood flow(CBF). Physically, astrocytes caused arteriolar dilation by release of vasoactive substances produced by either cytochrome P450 epoxygenase (P450) or cyclooxygenase (COX). The COX-2 expression change in AS after SAH, and the effects on morphologic change of microcirculation as well as and CBF change has not been investigated.PURPOSE: 1.To find the change of Ca²⁺and COX-2 in cultured astrocytes after incubated with hemolysate. 2. To investigate the morphologic changes of rat pial arterioles and rCBF with hemolysate. 3. To investigate intraparenchymal arterioles morphologic changes and tissue COX-2 expression using a rat cisterna magna autologous arterial blood injection model.METHODS: 1. Effects of hemolysate on intracellular free Ca²⁺ concentration and COX-2 expression in cultured astrocyte. Cultured AS were divided into 6groups. The cells were fixed with the corresponding incubated hemolysate, then the Ca²⁺ concentration were measured by spectrofluorometry using Fura-2/AM dying, and COX-2 expression observed by immunohistochemistry method. 2. Effects of hemolysate on pial microcirculation in rat . 36 male SD rats were divided into 6 groups. Animals were treated with either artificial CSF or hemolysate. Left craniotomy was performed and pial vasculature were exposed. Diameter changes were recorded by video record, and rCBF before and(1,5,10 min) after treatment were measured by laser Doppler. 3. Intraparenchymal arterioles morphologic changes and tissue COX-2 expression in a rat model of SAH. 49 male SD rats were divided into 7 groups. Autologous arterial blood were injected into cisterna magna in 0.1 ml/100g. Brains were embeded in paraffin, serial sections in 5μm carried on hematoxin eosin dying and COX-2 immunohistochemistry. Data are expressed as MEANS±SEM, Stata7.0 is used in statistical analysis.RESULTS: 1.The Ca²⁺concentration of treated group were from 268.23±10.36 to 365.3±18.14nmol/L, higher than control group. COX-2 over expressed in hemolysate treated groups. 2. Obviously narrow of pial arterioles appeared with the existence of hemolysate, diameters reduced from 38.9%⁵1%, venules were not affected. The initial rCBF were between 5.16±0.84 and 5.64±0.65, and rCBF reduction in 0,1,3,7d groups were 17.3%—52.7 %. Washed with artificial CSF, both the vessel diameter and rCBF were partly restored. 3. The arteriolar diameters in depth of 200um and 500um of 0, 1, 3, 7 and 14d were higher than control group. The COX-2 expression were weakly positive or negative in control and CSF groups, but positive or strongly positive in SAH groups, and this overexpression continued from 4h to 14d.CONCLUSIONS: l.The candidate for elevating the Ca²⁺+ concentration in hemolysate was OxyHb, there was linearity correlation between the OxyHb and Ca²⁺ in concentration ; COX-2 in AS over expressed due to high Ca²⁺ concentration. 2. OxyHb accounted for the constriction of rat pial arterioles, but had no effect on venules; the rCBF were decreased due to arteriolar constriction , arterioles in 30-50 μm might be the resistant vessels influencing the rCBF.